a) Field of the Invention
The invention is directed to a confocal microscope with a double-objective system comprising a first objective, which is provided for focussing light from an object illumination source onto an object point in an illumination beam path, and a second objective which is provided for collecting light that proceeds from the object point in an observation beam path, wherein the observation direction of the second objective is at an angle to the illumination direction of the first objective.
b) Description of the Related Art
A scanning microscope with a dual-objective system of the type mentioned above is known from DE 43 26 473 A1. In this known scanning microscope, collimated laser light is focussed through the first objective in the illumination beam path onto an object point. The second objective is arranged relative to the first objective in such a way that the optical axes of the two objectives are perpendicular to one another and the object point illuminated by the first objective lies in the principal focus of the second objective, so that the observation direction of the second objective is at a right angle to the illumination direction of the second objective. The observation light proceeding from the object point and collected by the second objective is focussed onto a pinhole diaphragm by a lens arranged following the second objective in the observation beam path, wherein a light detector measures the intensity of the light passing through the pinhole diaphragm. A corresponding image can then be generated from the measurement information of the light detector by scanning an object area.
Further, an incident illumination for stereo microscopes known from DE 94 08 066 U1 illuminates the object to be observed from the direction of the observation optics as far as possible. It is realized in such a way that the illumination arrangement forms a constructional unit with the microscope body and accordingly with the microscope objective acting as detector arrangement.
This illumination arrangement differs from the double-objective system according to the invention in particular in that it is not suitable for use in a confocal microscope and in that it illuminates as far as possible from the detection direction, whereas in the double-objective system according to the invention a right angle is realized as far as possible between the illumination direction and the detection direction.
The scanning microscope known from DE 43 26 473 A1 already has an extremely high resolution. This is achieved in particular in that the confocal volume detected in the in-focus or focal range is minimized due to the intersection of the optical axes of the illumination beam path and observation beam path at a right angle in the object point. When the illumination volume in the object area is considered in a rough approximation as an ellipsoid stretched along the illumination axis and the observation volume of the second objective in the object area is considered as an ellipsoid stretched along the optical axis of the second objective, a superposition of these ellipsoids in the focal range or in the object point region gives a correspondingly smaller confocal volume which substantially corresponds to the intersection volume of the illumination volume and observation volume. The smaller the confocal volume considered in this way, the better the resolution of the microscope.
An analogous manner of considering assumes that the intensity distribution of the object illumination light in the focal range of the first objective is described by the illumination point spread function, as it is called, and that the detection probability for the light proceeding from the focal range by means of the second objective when using a detection pinhole is described by the observation (detection) point spread function.
The resulting confocal point spread function KPVF of a confocal microscope is the product of the illumination point spread function and the observation point spread function. The more extensive the KPVF, the poorer the resolution of the microscope. As a result of the now virtually perpendicular arrangement of the illumination point spread function and the observation point spread function relative to one another, the comparatively large expanse of the illumination point spread function along the optical axis of the first objective is compensated by the small expanse of the illumination point spread function along this axis, so that an approximately equally good resolution is achieved along all three spatial axes. Therefore, a very good resolution capacity is achieved because of the illumination and observation beam paths which intersect at a right angle in the object point.
According to DE 43 26 473 A1, a special construction with complete separation of the observation beam path from the illumination beam path is provided for realizing the beam paths for illumination and observation which intersect in the object point. A special construction of this type requires a comparatively large amount of room in the two directions of the beam paths for illumination and observation at right angles to one another and is accordingly more difficult to manage than conventional scanning microscopes in which the illumination beam path and the observation beam path coincide and the focussing of the object illumination light and the collection of the observation light coming from the object point are effected through the same objective.